The relationship between tumour dosimetry, response, and overall survival in patients with unresectable Neuroendocrine Neoplasms (NEN) treated with 177Lu DOTATATE (LuTate).

Peptide Receptor Radionuclide Therapy (PRRT) delivers targeted radiation to Somatostatin Receptor (SSR) expressing Neuroendocrine Neoplasms (NEN). We sought to assess the predictive and prognostic implications of tumour dosimetry with respect to response by 68 Ga DOTATATE (GaTate) PET/CT molecular imaging tumour volume of SSR (MITVSSR) change and RECIST 1.1, and overall survival (OS). METHODS: Patients with gastro-entero-pancreatic (GEP) NEN who received LuTate followed by quantitative SPECT/CT (Q-SPECT/CT) the next day (Jul 2010 to Jan 2019) were retrospectively reviewed. Single time-point (STP) lesional dosimetry was performed for each cycle using population-based pharmacokinetic modelling. MITVSSR and RECIST 1.1 were measured at 3-months post PRRT. RESULTS: Median of 4 PRRT cycles were administered to 90 patients (range 2-5 cycles; mean 27.4 GBq cumulative activity; mean 7.6 GBq per cycle). 68% received at least one cycle with radiosensitising chemotherapy (RSC). RECIST 1.1 partial response was 24%, with 70% stable and 7% progressive disease. Cycle 1 radiation dose in measurable lesions was associated with local response (odds ratio 1.5 per 50 Gy [95% CI: 1.1-2.0], p = 0.002) when adjusted by tumour grade and RSC. Median change in MITVSSR was -63% (interquartile range -84 to -29), with no correlation with radiation dose to the most avid lesion on univariable or multivariant analyses (5.6 per 10 Gy [95% CI: -1.6, 12.8], p = 0.133). OS at 5-years was 68% (95% CI: 56-78%). Neither baseline MITVSSR (hazard ratio 1.1 [95% CI: 1.0, 1.2], p = 0.128) nor change in baseline MITVSSR (hazard ratio 1.0 [95% CI: 1.0, 1.1], p = 0.223) were associated with OS when adjusted by tumour grade and RSC but RSC was (95% CI: 0.2, 0.8, p = 0.012). CONCLUSION: Radiation dose to tumour during PRRT was predictive of radiologic response but not survival. Survival outcomes may relate to other biological factors. There was no evidence that MITVSSR change was associated with OS, but a larger study is needed.

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

A phantom for testing of 4D-CT for radiotherapy of small lesions.

PURPOSE: The use of time-resolved four-dimensional computed tomography (4D-CT) in radiotherapy requires strict quality assurance to ensure the accuracy of motion management protocols. The aim of this work was to design and test a phantom capable of large amplitude motion for use in 4D-CT, with particular interest in small lesions typical for stereotactic body radiotherapy. METHODS: The phantom of "see-saw" design is light weight, capable of including various sample materials and compatible with several surrogate marker signal acquisition systems. It is constructed of polymethylmethacrylate (Perspex) and its movement is controlled via a dc motor and drive wheel. It was tested using two CT scanners with different 4D acquisition methods: the Philips Brilliance Big Bore CT (helical scan, pressure belt) and a General Electric Discovery STE PET∕CT (axial scan, infrared marker). Amplitudes ranging from 1.5 to 6.0 cm and frequencies of up to 40 cycles per minute were used to study the effect of motion on image quality. Maximum intensity projections (MIPs), as well as average intensity projections (AIPs) of moving objects were investigated and their quality dependence on the number of phase reconstruction bins assessed. RESULTS: CT number discrepancies between moving and stationary objects were found to have no systematic dependence on amplitude, frequency, or specific interphase variability. MIP-delineated amplitudes of motion were found to match physical phantom amplitudes to within 2 mm for all motion scenarios tested. Objects undergoing large amplitude motions (>3.0 cm) were shown to cause artefacts in MIP and AIP projections when ten phase bins were assigned. This problem can be mitigated by increasing the number of phase bins in a 4D-CT scan. CONCLUSIONS: The phantom was found to be a suitable tool for evaluating the image quality of 4D-CT motion management technology, as well as providing a quality assurance tool for intercenter∕intervendor testing of commercial 4D-CT systems. When imaging objects with large amplitudes, the completeness criterion described here indicates the number of phase bins required to prevent missing data in MIPs and AIPs. This is most relevant for small lesions undergoing large motions.

A programmable motion phantom for quality assurance of motion management in radiotherapy.

A commercially available motion phantom (QUASAR, Modus Medical) was modified for programmable motion control with the aim of reproducing patient respiratory motion in one dimension in both the anterior-posterior and superior-inferior directions, as well as, providing controllable breath-hold and sinusoidal patterns for the testing of radiotherapy gating systems. In order to simulate realistic patient motion, the DC motor was replaced by a stepper motor. A separate 'chest-wall' motion platform was also designed to accommodate a variety of surrogate marker systems. The platform employs a second stepper motor that allows for the decoupling of the chest-wall and insert motion. The platform's accuracy was tested by replicating patient traces recorded with the Varian real-time position management (RPM) system and comparing the motion platform's recorded motion trace with the original patient data. Six lung cancer patient traces recorded with the RPM system were uploaded to the motion platform's in-house control software and subsequently replicated through the phantom motion platform. The phantom's motion profile was recorded with the RPM system and compared to the original patient data. Sinusoidal and breath-hold patterns were simulated with the motion platform and recorded with the RPM system to verify the systems potential for routine quality assurance of commercial radiotherapy gating systems. There was good correlation between replicated and actual patient data (P 0.003). Mean differences between the location of maxima in replicated and patient data-sets for six patients amounted to 0.034 cm with the corresponding minima mean equal to 0.010 cm. The upgraded motion phantom was found to replicate patient motion accurately as well as provide useful test patterns to aid in the quality assurance of motion management methods and technologies.

[Diaphragmatic rupture and right ipsilateral intercostal hernia in chronic cough]. / Rupture diaphragmatique et hernie intercostale droites homolatérales sur toux aiguë.

INTRODUCTION: We are reporting the case of a 64-year-old patient with chronic cough who has been diagnosed with an intercostal hernia with pleural and hepatic content associated with a diaphragmatic hernia of non-traumatic origin. CASE REPORT: The patient was treated for an acutely febrile cough with signs of respiratory distress. Thoracic scan showed an intercostal hernia containing an encysted hematoma and a right anterior diaphragmatic hernia with epiploic content. The COVID PCR was negative. This is one of the rare reported cases of intercostal hernia associated with a homolateral diaphragmatic rupture. Visceral and thoracic surgery enabled treatment of the two hernial orifices by raphy as well as omentectomy of the necrotic omentum ascending to the right pulmonary hilum. CONCLUSION: These two parietal complications of chronic cough should be considered in case of intercostal flap or acute respiratory distress. Surgery must then be carried out as a matter of urgency to reduce the content of the hernias and treat the musculoaponeurotic dehiscent orifices.

Motion effects on SUV and lesion volume in 3D and 4D PET scanning.

To assess the effect of lesion motion and respiration rate on Standardised Uptake Value (SUV) and the ability of 4D PET to restore any loss in SUV and distortion of lesion volume on two PET/CT systems. A Perspex phantom with four cylindrical reservoirs filled with (18)F-FDG was used in this study. The cylinders measured 5, 10, 15, and 20 mm in diameter. A GE Discovery STE8 (GE Medical Systems Milwaukee, WI) and a Siemens Biograph 64/40 (Siemens Medical Solutions, Erlangen, Germany) scanner was used to acquire a stationary un-gated PET scan of the phantom. Multiple 10 min list mode 4D PET scans were acquired using the Varian RPM on the GE camera and the Anzai Gating system on the Siemens camera. The phantom was scanned at five different respiratory rates and motion amplitudes in a sinusoidal fashion, 15 RPM/1 cm, 15 RPM/2 cm, 15 RPM/4 cm, 30 RPM/2 cm and 7.5 RPM/2 cm (RPM-respirations per minute). Each scan was reconstructed into ten bins and as an un-gated static image. The SUVmax, SUVmean and volume were measured for all four reservoirs using Siemens TrueD analysis software. With increasing lesion movement the SUVmax and SUVmean decreased and the volume increased with the SUVmax in the smallest lesion underestimated by up to a factor of four. The SUVmax, SUVmean and volume were mostly recovered using 4D imaging regardless of amount of lesion displacement. The larger lesions showed better count recovery and volume correction than the smaller lesions. The respiratory rate had no effect of SUV or volume. Un-gated imaging of moving lesions decreases apparent SUV in small lesions significantly and overestimates volumes. 4D PET scanning recovers most of the apparent loss in SUV and distortion of volumes.

Effects of hydrodynamic pressure processing on the marination and meat quality of turkey breasts.

The effects of hydrodynamic pressure processing (HDP) on marination and meat quality characteristics of turkey breasts were investigated. Breast muscles from 45 turkey hens were removed from the carcasses within 30 min postmortem. From each bird, the breast from one side was treated with HDP and the other side served as a nontreated control. Breasts were then marinated in either 15 or 30% brine (water, salt, and phosphate) based on muscle weight with vacuum tumbling for 30 min or nonmarinated. The control and HDP-treated breasts from each bird received the same marination treatment. Brine uptake, processing yield, and cooking loss were measured as processing characteristics and texture, color, and expressible moisture were measured to document changes in meat quality. Hydrodynamic pressure processing increased (P < 0.001) brine uptake after 10 and 30 min of marination and increased (P < 0.001) processing yield compared with controls. The HDP-induced improvements in these processing characteristics were augmented at 30% brine levels compared with 15% brine. Cooking loss was lower (P < 0.001) in marinated breasts compared with nonmarinated samples. Hydrodynamic pressure processing decreased (P < 0.0001) Warner-Bratzler shear force and significantly influenced texture profile parameters, resulting in reduced hardness but increased cohesiveness and springiness compared with controls at both marination levels. Hydrodynamic pressure processing did not influence color (L*, a*, and b*) or expressible moisture values compared with controls at either marination level. Marinated samples (15 and 30% brine levels) had lower (P < 0.001) Warner-Bratzler shear force values and lower (P < 0.05) hardness, cohesiveness, and chewiness values compared with nonmarinated samples. Data from this study suggest that HDP enhances brine absorption, increases processing yield, and improves texture characteristics in marinated turkey breasts.

Large odd-numbered carbon clusters from fullerene-ozone reactions.

The odd-numbered carbon clusters C(119), C(129), and C(139) have been observed in the mass spectra of toluene extracts of fullerene soots and of the products of ozone-fullerene reactions. Specifically, ozone-C(60) reactions yield C(119), ozone-C(70) reactions yield C(139), and ozone-(C(60)/C(70)) reactions produce C(119), C(129), and C(139). These unexpected species correspond to dimers of C(60), C(60)/C(70), and C(70), respectively, less one carbon atom, and are stable gas-phase ions with behavior similar to that of fullerenes. The results suggest a new route to functionalization and derivatization of fullerenes through controlled ozone-catalyzed cage-opening reactions.

The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.

Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

27Al MQMAS of the δ-Al13-Keggin.

One-dimensional 27Al, 23Na Magic-Angle-Spinning (MAS) NMR and 27Al Multiple-Quantum Magic-Angle-Spinning NMR (MQMAS) measurements are reported for the δ-isomer of the Al13 Keggin structure at high spinning speed and 14.1 T field. Values for the CQ and η parameters are on the same scale as those seen in other isomers of the Al13 structure. Density functional theory (DFT) calculations are performed for comparison to the experimental fits using the B3PW91/6-31+G* and PBE0/6-31+G* levels of theory, with the Polarizable Continuum Model (PCM).

Modification of the Perinatal PTSD Questionnaire to enhance clinical utility.

OBJECTIVE: To enhance the clinical utility of the Perinatal Post-Traumatic Stress Disorder (PTSD) Questionnaire (PPQ), the current study sought to refine the measure by changing the item response options from dichotomous choices to a likert scale format. STUDY DESIGN: Using a convergent/divergent validity design and two data sources (traditional survey and World Wide Web), 58 high-risk and 86 low-risk mothers answered four questionnaires. RESULTS: Principal components analysis of items on the modified PPQ revealed three components conceptually similar to the diagnostic criterion associated with PTSD. In addition, convergent and divergent validity of the modified measure was supported. The clinical utility of the modified PPQ was established with a strong positive likelihood ratio. CONCLUSION: The modified PPQ is a useful clinical tool for identifying mothers experiencing significant emotional distress during the postnatal period so they may be referred for mental health services.

Molecular basis of GM1 gangliosidosis and Morquio disease, type B. Structure-function studies of lysosomal beta-galactosidase and the non-lysosomal beta-galactosidase-like protein.

GM1 gangliosidosis and Morquio B disease are distinct disorders both clinically and biochemically yet they arise from the same beta-galactosidase enzyme deficiency. On the other hand, galactosialidosis and sialidosis share common clinical and biochemical features, yet they arise from two separate enzyme deficiencies, namely, protective protein/cathepsin A and neuraminidase, respectively. However distinct, in practice these disorders overlap both clinically and biochemically so that easy discrimination between them is sometimes difficult. The principle reason for this may be found in the fact that these three enzymes form a unique complex in lysosomes that is required for their stability and posttranslational processing. In this review, I focus mainly on the primary and secondary beta-galactosidase deficiency states and offer some hypotheses to account for differences between GM1 gangliosidosis and Morquio B disease.

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.

The biochemistry and clinical features of galactosialidosis.

Galactosialidosis is a heterogeneous disorder that is manifested in infantile, late infantile, juvenile/adult, and atypical forms. In every instance the primary defect is in the ability of protective protein to associate with beta-galactosidase and neuraminidase to protect them from intralysosomal proteolysis. The protective protein is in reality a serine protease that displays both cathepsin A and C-terminal deamidase activity. We summarize the major clinical features of each form, and the range of storage products accumulated. The concept of an intralysosomal complex containing beta-galactosidase and neuraminidase in addition to protective protein seems irrefutable but major gaps exist in our understanding of how the complex is formed and in what subcellular organelles, how it is sustained, and the protein domains contributed by the constituent enzymes that play a pivotal role in this process.

Classification of disorders of GM2 ganglioside hydrolysis using 3H-GM2 as substrate.

Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibroblast extracts in the presence of no additives, sodium taurocholate, and GM2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated GM2 while fibroblasts from patients with GM2 gangliosidosis did not. The half life of GM2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed GM2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of GM2 hydrolysis by normal fibroblast extracts were increased 5-16-fold, fibroblast extracts from AB and B1 variant patients hydrolyzed GM2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed GM2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed GM2 at about 10% of normal rates. In the presence of 1 microgram of GM2 activator, rates of GM2 hydrolysis by normal fibroblast extracts were increased 8-25-fold, fibroblast extracts from a patient with the AB variant hydrolyzed GM2 at normal rates, and cell extracts from other variants of GM2 gangliosidosis did not hydrolyze GM2. The results suggest that measuring the persistence of 3H-GM2 in tissue culture over time will detect any variant of GM2 gangliosidosis and may be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of 3H-GM2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator.

Concanavalin A promotes the uptake of lysosomal hydrolases by human fibroblasts.

Human placental hexosaminidase B and beta-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4 degrees C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37 degrees C is refractory to haptens treatment. These results are interpreted as an initial binding of enzyme to concanvalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.

Studies on the activation of the enzymatic hydrolysis of sphingomyelin liposomes.

Two pH optima were observed for the hydrolysis of sphingomyelin liposomes by brain and fibroblast extracts; one at pH 4.2-4.5, the other at pH 7-8. The proportion of the acidic activity in fibroblasts was affected greatly by the culturing conditions. Both the acidic and neutral enzyme activities were deficient in Niemann-Pick Type A fibroblasts, suggesting that both were genetically related. Partially purified activators from normal as well as Gaucher disease spleen stimulated the hydrolysis of sphingomyelin, at both pH values, by fibroblast and brain extracts. After further purification by DE-52 and Sephacryl 200 column chromatography the Gaucher activator retained its ability to stimulate sphingomyelinase and was active as well towards beta-glucocerebrosidase and beta-galactocerebrosidase.

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U